ABSTRACT Malaria kills an estimated 1-2 million people (mostly children) every year. For transmission to occur, Plasmodium, the causative agent of malaria, has to complete a complex developmental cycle in the mosquito. Only a small proportion of the parasites survive the entire cycle. Thus, the mosquito is a potential weak link that can be exploited for disease control. Invasion of the mosquito midgut by Plasmodium ookinetes is a crucial step, yet little is known about the molecular mechanisms that operate at this stage. We made two unexpected observations during the current grant period: 1) The surface of Plasmodium ookinetes (the form that invades the midgut) is lined with an enolase-like protein and 2) ookinetes can invade the midgut by more than one pathway, one that can be blocked by the SM1 peptide and another that cannot. One aim of this proposal is to investigate, at the molecular level, the mechanism of midgut invasion. Our first aim will address the following working hypothesis. Enolase expressed on the surface of midgut ookinetes captures plasminogen from the surrounding blood meal. A mosquito type II annexin on the surface of the midgut epithelium binds to both tissue type plasminogen activator (tPA) from the blood meal and to ookinete surface enolase. We hypothesize that this bridge facilitates both ookinete docking to the surface of the midgut epithelium and tPA activation of plasminogen into plasmin (a protease). The combination of these two separate but intimately entwined events results in successul midgut invasion. Our second aim is to identify P. berghei ookinete genes that are responsible for the different invasion pathways. This is the first comprehensive study of the mechanisms of Plasmodium invasion of the midgut epithelium. Knowledge generated by these studies may have important implications for the development of multivalent transmission-blocking vaccines.